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39774.

Cytochrome P450 and the Metabolism

and Bioactivation of Arachidonic Acid

and Eicosanoids

Jorge H. Capdevila, Vijaykumar R. Holla, and John R. Faick

Introduction

The convergence of important advances in the

identification of several lipid-derived mediators as

inter- and intracellular signaling molecules, and in

the biochemistry of oxidative lipid metabolism,

has focused interest in the functional roles of path-

ways responsible for their formation, and the

physiological significance of their products.

Among these, the studies of the enzymes of the

arachidonic acid (AA) cascade, consisting of

prostaglandin H2 synthase^'^, lipoxygenases^, and

cytochrome

P450'^'

constitute a premier example

of the biological importance of these reactions and

of their products (eicosanoids). Studies of

the

last

two decades, have implicated the enzymes of the

AA cascade in the pathophysiology of diseases

such as hypertension, diabetes, and cancer, and

some of these enzymes serve as molecular targets

for drugs of extensive use in clinical medicine,

including many nonsteroidal anti-inflanmiatory,

antipyretic, and anti-asthmatic drugs^~^. The bio-

logical and signaling properties of eicosanoids are

derived from the enzymatic, regio-, and stereo-

selective oxygenation of AA, a rather simple

molecular template. While the informational

content stored in the AA metabolites, is limited

compared to that contained in complex informa-

tional molecules such as proteins or nucleic acids,

low energy cost, versatility, and rapid turnover,

makes them efficient on/off molecular switches

for rapid and efficient intra- or intercellular sig-

naling. Metabolism by prostaglandin H2 synthase

generates a cyclic endoperoxide, prostaglandin

H2 (PGH2) that serves as the precursor for the

formation of prostaglandins, prostacyclin, and

thromboxanes^' ^. Metabolism by lipoxygenases

leads to the formation of several regioisomeric

hydroperoxides, the precursors of leukotrienes,

regioisomeric cis/trans conjugated hydroxye-

icosatetraenoic acids (HETEs), lipoxins, and hep-

oxilins^. Metabolism by microsomal cytochrome

P450s (P450s) generates several hydroxy- and

epoxy-AA derivatives'^'

The reactions catalyzed

by prostaglandin H2 synthase and lipoxygenases

are mechanistically similar to those of the free-

radical-mediated autooxidation of polyunsaturated

fatty acids in that they are initiated by hydrogen

atom abstraction from a bis-allylic methylene

carbon, followed by coupling of the resulting car-

bon radical to groimd state molecular oxygen. The

kinetics, regiochemistry, and chirality of these

Jorge H. Capdevila • Departments of Medicine and Biochemistry, Vanderbilt University Medical School,

Nashville, TN. Vijaykumar R. Holla • Department of Medicine, Vanderbilt University Medical School,

Nashville,

TN.

John R. FaIck • Department of

Biochemistry,

Southwestern Medical Center, Dallas, TX.

Cytochrome

P450:

Structure,

Mechanism,

and

Biochemistry,

3e, edited by Paul R. Ortiz de Montellano

Kluwer

Academic

/ Plenum Publishers,

New

York,

2005.

531

532

Jorge H. Capdevila et al.

reactions are under strict enzymatic control. In

contradistinction to the c)^ochrome P450-catalyzed,

redox coupled, activation of molecular oxygen and

delivery to ground state carbon, prostaglandin H2

synthase and the lipoxygenases are typical dioxyge-

nases that catalyze substrate carbon activation

instead of oxygen activation.

It is apparent from the literature that the P450

gene superfamily of hemoproteins is, as a group,

one of the most intensively studied enzyme sys-

tems and yet our knowledge of their endogenous

metabolic or physiological roles remains limited.

This is partly due to the complexity of mammalian

P450 isoforms, and the wide structural diversity

of substrates known to be metabolized by these

proteins. This catalytic versatility pointed to, and

served as the basis for, many of the documented

roles for P450 in the metabolism of foreign chem-

icals,

and has contributed to establish its toxico-

logical and pharmacological importance. In the

last few years, there has been an increasing inter-

est in the understanding of the physiological sig-

nificance of the P450 enzyme system, and its

role(s) in the metabolism of endogenous sub-

strates. In this regard, the studies of the P450

branch of the AA cascade have provided a new

focus to these efforts, and far-reaching results

from several laboratories are generating new par-

adigms in fatty acid metabolism, as well as in cell

and organ physiology^^^.

The studies of the role of P450 in the metabo-

lism and bioactivation of AA were initiated in 1981

with the demonstration that liver and kidney

microsomal fractions, as well as purified P450

isoforms^

^"^^

actively catalyzed the NADPH-

dependent, oxidative metabolism of AA to prod-

ucts that were different from prostanoids and

leukotrienes"'^. The widely documented physio-

logical importance of AA suggested that these

observations were unique and likely to be func-

tionally significant, and led to the rapid structural

characterization of most P450-eicosanoids, their

chemical synthesis, and subsequent biological

evaluation"^'^. Interest in these novel P450 reac-

tions was stimulated by: (a) the initial demonstra-

tion that some of the products displayed potent

biological activities, including the inhibition of

Na^ reabsorption in the distal nephron^^, (b) the

documentation of

P450

participation in the in vivo

metabolism of endogenous AA

pools^^,

and (c) the

proposal of a role for these enzymes in the

pathophysiology of genetically controlled experi-

mental hypertension^'

These earlier studies estab-

lished P450 AA monooxygenation as a formal

metabolic pathway, P450 as an endogenous mem-

ber of the AA metabolic cascade, and more impor-

tantly, suggested functional roles for this enzyme in

the bioactivation of the fatty acid and thus, in cell

and organ physiology. Many of the biological activ-

ities attributed to the P450-derived eicosanoids,

as well as the potential physiological importance of

these reactions, have been reviewed^"^^.

Prior to the demonstration of AA metabolism

by P450, several groups demonstrated the role of

microsomal P450s in the (o/w-l hydroxylation of

prostanoids^^"^^ and, more recently, leukotrienes^^.

Most of these reactions are considered to be

involved in eicosanoid catabolism and excretion,

but their potential relevance in eicosanoid bioacti-

vation or inactivation, and/or in the control of

organ/cell eicosanoid levels has only begun to be

explored. We will first discuss the role of P450

in the metabolism of eicosanoids, and then con-

centrate on the studies of its role in AA metabolism

and bioactivation.

Metabolism of Eicosanoids

During the metabolism of eicosanoids,

depending on the nature of the oxygenated sub-

strate, P450 catalyzes both NADPH-dependent

and -independent reactions. This differential

requirement for NADPH-mediated changes in the

redox state of the heme-iron illustrates the marked

differences in oxygen chemistries for these reac-

tions,

that is, the isomerization of AA peroxides,

vs the more demanding activation and delivery of

a reactive form of atomic oxygen to ground state

carbon-hydrogen bonds.

2.1.

NADPH-lndependent

Reactions

P450 catalyzes the isomerization of a variety

of fatty acid hydroperoxides, including 15-hydro-

peroxyeicosatetraenoic acid (15-HPETE)^^' ^^,

and of the prostaglandin H2 (PGH2) endoperox-

ide^^.

A distinctive feature of some P450 fatty acid

peroxide isomerases is their inability to accept

electrons from NADPH and to activate molecular

Cytochrome P450 and

the

Metabolism

AA and Eicosanoids

533

oxygen^ ^"^^. Moreover, while

all

these enzymes

possess

heme-thiolate prosthetic group, their

overall homology

other members

the P450

gene superfamily

limited

and

suggests

early

evolutionary functional specialization^^"^^.

The

mechanism

which the hemoprotein cleaves

the

peroxide oxygen-oxygen bond, that

is,

homolytic

or heterolytic scission, plays

decisive role

determining

the

catalytic outcome

these reac-

tions

and is

highly dependent

on the

nature

the P450 isoform,

the

chemical properties

the

organic peroxide,

and the

nature

of the

oxygen

acceptor^^"^^'

^^.

A homolytic pathway was proposed

for

the

formation

of 11- and

13-hydroxy-14,

15-epoxyeicosatrienoic acids (EETs) from

15-

HPETE

by rat

liver microsomes'^. Prostacyclin

and thromboxane synthases are P450-like proteins

containing

heme-thiolate prosthetic group'^~'^.

The heterolytic cleavage

PGH2

and an

oxygen

atom transfer

oxenoid mechanism

has

been

proposed to account for the P450-catalyzed forma-

tion

prostacyclin (PGI2)

and

thromboxane

(TXA2)'^.

The

participation

P450s

in the

bio-

synthesis

these important mediators

endothe-

lial cell

and

platelet function

was one of

the first

demonstrations

role

for

this enzyme system

vascular

biology.

However, its pharmacological and

clinical implications remain to be fully explored.

terminal (C20

co-carbon)

penultimate carbon

atoms (C

(0-1

carbon). However,

the

epoxida-

tion

infused PGI2

by a

perfused kidney prepa-

ration'^,

and the

metabolism

of 5,6- and

8,9-EET

prostaglandin

synthase, were

described several years ago'^'

^^.

The former leads

variety

5,6-oxygenated prostanoids'^.

Oxidation

of the

latter

was

stereodependent,

that

is,

8(5),9(i?)-EET formed ll(i?)-hydroxy-

8(-S),9(i?)-epoxyeicosatrienoic acid exclusively,

whereas

the

8(/?),9(*S)-enantiomer formed both

Cjj

and Cj5

hydroxylated metabolites^^.

detailed study

of the

secondary metabolism

12(i?)-HETE

and

14,15-EET

P450

has

been

reported^ ^'

^'. The

efficient

w/w-l

oxidation

EETs

by rat

CYP4A isoforms

to the

correspon-

ding regioisomeric epoxy-alcohols

rates com-

parable

those observed with lauric acid,

prototype substrate

for

these enzymes,

was

pub-

lished recently^^.

One of

these metabolites,

the

o)-alcohol

14,15-EET,

was

shown

bind

and

activate

the

peroxisomal proliferator activated

receptor alpha (PPAR^) type of nuclear receptor^ ^.

important

note that, with

the

exception

the (o/o)-! hydroxylation

prostanoids

and

leukotrienes, none of the transformations described

above

has

been shown

occur

vivo from

endogenous precursors.

2.2.

NADPH-Dependent

Reactions

P450 plays

important role

in the

NADPH-

dependent metabolism

several bioactive oxy-

genated eicosanoids^^'^. These reactions

are of

importance

that they:

(a)

increase eicosanoid

structural diversity

and,

hence, modify informa-

tional content,

(b) may

alter

the

pharmacological

profile

of the

substrate,

and (c) may

participate

the

regulation

steady state and/or stimulated

levels

physiologically relevant molecules. While

these oxidations were generally viewed as catabolic,

that

is,

yielding

attenuated biological activity,

recent studies indicate that some w-oxidized pro-

stanoids show unique and potent biological proper-

^jgg6-io However,

most cases

the

sequence

steps leading

to w/w-l

oxidized prostanoids from

endogenous AA pools remains

be clarified.

Typically,

the

P450-dependent metabolism

eicosanoids results

in the

hydroxylation

their

2.2.1.

ct)/(o-1 Oxidation

Prostanoids

Since

the

initial report

vivo

w-l

hydroxy-

lation

prostanoids

1966^^'

^^, w- and o)-l

hydroxylation

has

become

recognized route

prostanoid metabolism. Early studies of prostanoid

o)/o)-l hydroxylation demonstrated these reactions

were NADPH-dependent, localized

to the

endo-

plasmic reticulum^^'

^^,

and

catalyzed

microso-

mal P450

(ref

[36]). Incubations with purified

enzymes

recombinant P450s showed that most

of these reactions were catalyzed

members

the 4 gene family of

P450s'^'

^^^^.

In general, while

CYP4F isoforms are more active in the metabolism

of eicosanoids than fatty acids, the opposite appears

true

for

most CYP4A isoforms'^'

^^.

Approximately

CYP4F isoforms have been

identified

rats (4F1, 4F4, 4F5, and 4F6)4^ mice

(4fl3,

4fl4,

4fl5,

4fl6, 4fl7, 4f37,

409, and

4f40)45,

and

humans (4F2, 4F3, 4F8, 4F11, 4F12,

534

Jorge H. Capdevila ef a/.

Table 11.1. Metabolism of Fatty Acids and Prostanoids by

Cytochrome P450 4A Isoforms

4A isoform Species

Enzymatic activities"

4A1

4A2

4A3

4A8

4A4

4A5

4A6

4A7

4al0

4al2

4al4

4A11

4a22

Rat

Rabbit

Mouse

Human

(o-oxidation of laurate and arachidonate

co/w-l oxidation of laurate and arachidonate

(o/o)-!

oxidation of laurate and arachidonate

(o/w-l oxidation of laurate and arachidonate

(rt-oxidation of palmitate, arachidonate, and of

prostaglandins A, E, D, and F2^

co/(x)-l oxidation of laurate and palmitate,

some a)-oxidation of PGAj and arachidonate

w-oxidation of laurate, palmitate, and

arachidonate. Low PGAj w-oxidation

(o-oxidation of

laurate,

palmitate, arachidonate,

and

PGAj,

inactive toward PGE2

(O-oxidation of laurate

co/o)-] oxidation of laurate and arachidonate

(o/o)-1 oxidation of laurate

(o/to-1 oxidation of laurate and arachidonate

Unknown

''Compiled from references [4]-[10], [17]-[20], [36H44], [46]-[52].

and 4F22)'^^. On the other hand, approximately 11

CYP4A isoforms have been cloned and/or isolated

and purified from rats (4A1,4A2,4A3, and

4A8)'^^,

mouse (4a

10,

12,

and 4a

14)"^^,

and rabbits (4A4,

4A5,

4A6, and 4A7)'^^. In stark contrast with the

known multiplicity of rodent CYP4A and of rodent

and human CYP4F isoforms, only two highly

homologous CYP4A genes, CYP4A11 and

CYP4A22, have been identified in humans"*^"^^.

Most CYP4A enzymes that have been character-

ized enzymatically, are either inactive toward

prostanoids or catalyze their (o- or

o)/o)-1

hydroxy-

lation at rates that are generally substantially lower

than fatty acid hydroxylation (Table 11.1)^^' ^^~^'

^^~^K

A special case is that of rabbit lung CYP4A4,

an isoform induced during pregnancy^^' ^^' ^^' ^^

and active in the co-hydroxylation of several

prostanoids, including PGE2 (Table 11.1)^^'

^^.

with AA, none of the CYP4A isoforms character-

ized to date is selective for the a)-l carbon of

prostanoids (Table

11.1)^^'

^^^^'

^^~^^.

A more spe-

cific role for CYP4F isoforms as predominantly

prostanoid and eicosanoid w/co-l hydroxylases has

emerged during the last few years^^. The cDNA

coding for CYP4F8 was cloned from human semi-

nal vesicles, and the recombinant protein was shown

to catalyze the o)-l hydroxylation of

PGH2,

the pre-

cursor of all prostanoids"^^. Based on its catalytic

activity, and its high levels of expression in the

seminal vesicles it was proposed that CYP4F8 is

involved in the formation of 19-hydroxy-PGE2,

present at high concentrations in human seminal

fluid34,

CYP4F12 is a regioselective AA a)-3

hydroxylase^^ but, it is also active in the hydro-

xylation of prostanoids and several prostanoid

analogs^^.

2.2.2.

a>/co-1 Oxidation of

Leukotrienes and Other

Eicosanoids

The (O-oxidation of leukotriene B4 (LTB^), an

important proinflammatory mediator^^' ^^, has

been documented in whole animals, isolated cells,

and subcellular fractions^^'

^^^o,

and shown to be

catalyzed by a unique P450 isoform, distinct from

those involved in fatty acid and prostanoid metab-

olism^^'

^^~^^.

Soon after, the cDNA coding for

CYP4F3 was cloned and expressed, and recombi-

nant CYP4F3 shown to catalyze the o)-oxidation

of LTB4 with a K^ of 0.71 luiM^i'

^2.

A role for